Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F17/00—Metallocenes
  • C07F17/02—Metallocenes of metals of Groups 8, 9 or 10 of the Periodic Table
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

• the invention relates to electrochemical detection methods. More especially, the invention relates to electrochemical assays, to electrochemically active labels suitable for use in electrochemical detection methods, and to their use.
• WO03/074731 discloses a method of probing for a nucleic acid.
• a nucleic acid solution is contacted with an oligonucleotide probe with an electrochemically active marker.
• the probe is caused to at least partially hybridise with any complementary target sequence which may be present in the nucleic acid solution.
• information is electrochemically determined relating to the marker.
• Compounds for use in the method are also disclosed.
• WO2005/005657 discloses a method of detecting protease activity in which a sample solution is contacted with a protease substrate with an electrochemically active marker, providing conditions under which any protease which may be present in the sample may degrade the protease substrate and information relating to the electrochemically active marker is electrochemically determined. Certain novel compounds for use in the process were also disclosed.
• WO2012/085591 describes certain diferrocenyl compounds for use as electrochemical labels.
• labels that enable detection of the presence in small concentrations of biological substrates or indicators, for example, nucleic acids (in isolated form or in the form of larger molecules, for example, natural or synthetic oligonucleotides), or amino acids (in isolated form or in the form of larger molecules, for example, natural or synthetic peptides).
• nucleic acids in isolated form or in the form of larger molecules, for example, natural or synthetic oligonucleotides
• amino acids in isolated form or in the form of larger molecules, for example, natural or synthetic peptides.
• new labels with different oxidation potentials and/or with different chemical or physical properties thereby widening the range of possible assays available and increasing the scope for the development of multiplex reactions.
• Palladium catalysts based on these ligands efficiently promote asymmetric allylic alkylation of 1,3-diphenylallyl acetate with in situ generated dimethyl malonate anion to give the C-alkylated product with ees up to 93% at room temperature .”
• WO 2011/073675 discloses "A method of detecting genetic material deriving from Chlamydia trachomatis comprising detection of a specified nucleic acid sequence, optionally using specific primers and probes and optionally in combination with the detection of genetic material deriving from Pectobacterium atrosepticum as an internal control; and related products and kits.”
• the invention provides a compound according to general formula I in which:
• the invention also provides use as a label in an electrochemical assay of a compound of general formula I: in which:
• the compounds used in accordance with the invention have been found to be effective labels suitable for use in electrochemical assays.
• the compounds may be used to form labelled substrates.
• Molecules of interest as substrates that may be labelled include, but are not limited to: amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates and derivatives or synthetic analogs of any of those molecules.
• the labelling compounds of general formula I and labelled molecules including labels derivable from the labelling compounds are potentially useful in electrochemical techniques in which their electrochemical characteristics can be utilized to derive information about the labels or their environment.
• the compounds of the invention may find use in a method as described in WO03/074731 or in a method as described in WO2005/005657 .
• the labelling compounds of the invention and the labelled substrates derived therefrom offer characteristics which make them useful complements to previously known labelling compounds, permitting a wider spectrum of applications, for example, offering additional opportunities for avoidance of conditions under which measurement potential may be compromised by interference with impurities that may be present and/or offering differing electrochemical potential values and/or allowing more greater flexibility in multiplex assays.
• a number of the compounds and the corresponding labelled substrates have relatively high electro potential values, as illustrated in particular by Example 4 below.
• X is a C1 to C6 alkylene chain which is optionally interrupted by -O- , -S- , or -NR 5 -, in which R 5 represents hydrogen or C1 to C6 alkyl
• Y is a C1 to C6 alkylene chain which is optionally interrupted by -O-, -S-, or -NR 5 -, in which R 5 represents hydrogen or C1 to C6 alkyl
• Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by -O-, -S-, cycloalkyl, -CO-, -CONR 1 -, -NR 1 CO- or -NR 1 - in which R 1 represents hydrogen or C1 to C4 alkyl.
• X represents C1- to C6-alkylene optionally interrupted by oxygen
• Y represents C1 to C6-alkylene optionally interrupted by oxygen
• Z represents C1 to C8 alkylene optionally interrupted by oxygen.
• X, Y and Z can be represented by the formula (CH 2 ) a -O-(CH 2 ) b wherein a ⁇ 0 and b ⁇ 0.
• a + b 1-6.
• X is preferably -(CH 2 ) x - in which x is from 1 to 6, preferably 1 to 4, especially 1 or 2; or C1 to C6-alkylene interrupted by oxygen, for example -(CH 2 ) 3 -O-CH 2 -, -(CH 2 ) 2 -O-(CH 2 ) 2 -, or -CH 2 -O-(CH 2 ) 3 -.
• Y is preferably -(CH 2 ) y - in which y is from 1 to 6, preferably 1 to 4, especially 1 or 2; or C1 to C6-alkylene interrupted by oxygen, for example -(CH 2 ) 3 -O-CH 2 -, -(CH 2 ) 2 -O-(CH 2 ) 2 - or -CH 2 -O-(CH 2 ) 3 -.
• X and Y are the same.
• Fc and Fc' are the same and X and Y are the same.
• Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by -O- , -S- or -NR 1 - in which R 1 represents hydrogen or C1 to C4 alkyl.
• Z is -(CH 2 ) z - in which z is from 1 to 8, with z preferably representing from 1 to 6, especially from 2 to 6; or is C1 to C8 alkylene interrupted by oxygen, for example - (CH 2 ) 2 -O-(CH 2 ) 3 - or -(CH 2 ) 3 -O-(CH 2 ) 2 -.
• X is -(CH 2 ) x - in which x is 1 or 2; Y is - (CH 2 ) y - in which y is 1 or 2; and Z is -(CH 2 ) z - in which z is from 1 to 8.
• R 5 preferably represents hydrogen or C1 to C4 alkyl, more preferably hydrogen.
• the invention provides use, as an electrochemical label, of a compound of the general formula II: in which:
• x and y are each equal to 1.
• ferrocenyl moieties are the same, and it is therefore preferred that Fc and Fc' carry the same substituents in the same positions.
• substrate is used throughout the remainder of this document to include both naturally occurring substrates and synthetic substrates, and references herein to amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, or carbohydrates, are to be understood as referring to naturally occurring or synthetic amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, or carbohydrates. Substrates can also be polypeptides. Synthetic substrates include synthetic analogues of naturally occurring substrates. Substrates include single nucleotides and single amino acids.
• a single amino acid may be regarded as a substrate because, although it lacks an internal bond capable of being cleaved by a protease enzyme, such a bond may be formed through the attachment of a marker.
• those derivatives may be naturally occurring derivatives or synthetic derivatives of the substrate.
• the invention provides a method of detecting a chemical entity using a compound according to the invention.
• Use in an electrochemical assay according to the invention may be for example in an assay for detecting an electrochemically labelled substrate.
• the electrochemical assay may for example be an assay for determination of the amount of an electrochemically labelled substrate.
• the assay may advantageously be for detecting or determining the amount of a labelled substrate wherein the labelled substrate is selected from amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, and carbohydrates.
• the assay is for detecting or determining the amount of a labelled substrate in which the labelled substrate is selected from nucleotides, nucleosides, oligonucleotides, and polynucleotides. In another advantageous embodiment, the assay is for detecting or determining the amount of a labelled substrate in which the labelled substrate is selected from amino acids, peptides, and proteins.
• the label may be functionalised by addition of a functionalising group.
• the invention further provides functionalised derivatives comprising a moiety derivable from the compounds of the invention attached to a functionalising group suitable for enhancing attachment to a substrate.
• the invention also provides a method for manufacturing a functionalized labelling compound comprising an electrochemically active label moiety suitable for use in an electrochemical assay, wherein the method comprises the step of reacting a compound of general formula I: with a functionalising compound to obtain a functionalised labelling compound comprising a succinimidyl ester group, phosphoramidite group, maleimide group, biotin or azide group; wherein the compound of general formula I:
• the invention provides a method for the manufacture of a labelled substrate, comprising reacting the functionalised labelling compound as defined above with a substrate to form a labelled substrate, wherein the substrate is
• the invention moreover provides a functionalised labelling compound suitable for use in the manufacture of a labelled substrate, wherein the functionalised labelling compound is a functionalised derivate of a compound of general formula I, comprising a moiety selected from succinimidyl ester groups, phosphoramidite groups, maleimide groups, biotin and azide groups.
• the invention also provides a labelled substrate suitable for use in an electrochemical assay, wherein the substrate is labelled with a compound of general formula I or a functionalised labelling compound according to the method described above, and wherein.
• alkyl are to straight- or branched-chain alkyl groups preferably having from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms; or to cycloalkyl groups.
• Alkyl or cycloalkyl groups may be optionally interrupted by a heteroatom selected from O, S and N and/or optionally have one or more substituents.
• Illustrative alkyl groups include, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl.
• cycloalkyl are to cycloalkyl groups having up to eight, preferably up to six, ring carbon atoms. Cycloalkyl groups may optionally include one or more heteroatoms. Illustrative cycloalkyl groups include, for example, cyclohexyl and heterocyclic groups such as piperidinyl and morpholinyl.
• alkenyl are to straight- or branched-chain alkenyl groups preferably having from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms, optionally having one or more substituents.
• Illustrative alkenyl groups include, for example, ethenyl, propenyl, butenyl.
• haloalkyl is used herein, except where the contrary is indicated, to refer to alkyl groups having one or more halogen atoms present as substituents, said one or more halogen atoms being selected from fluorine, chlorine, bromine and iodine.
• sulfur-containing group will be understood as including, without limitation, substituent groups including an -S(O)2 - moiety (referred to herein as “sulfonyl”), an -S(O)- moiety (referred to herein as “sulfinyl”) or an -S- moiety (referred to herein as "sulfenyl”).
• Preferred sulfur-containing groups that may be present as substituents on the ferrocenyl rings in accordance with the invention are those in which the sulfur atom is directly bonded to a ring carbon.
• phosphorus-containing group will be understood as including, without limitation, substituent groups including those based on phosphines or phosphine oxides, more particularly phosphanyl (>P-) and phosphinyl (>P(O)-) groups.
• Preferred phosphorus-containing groups that may be present as substituents on the ferrocenyl rings in accordance with the invention are those in which the phosphorus atom is directly bonded to a ring carbon.
• heteroaryl are to be understood as including any single or fused aromatic moiety including one or more heteroatoms, the heteroatoms preferably being selected from oxygen, sulphur and nitrogen. Where more than one heteroatom is present the heteroatoms may be the same or different, each advantageously being independently selected from oxygen, sulphur and nitrogen.
• Illustrative heteroaryl groups include without limitation furanyl, imidazolyl, thiazolyl.
• aryl are to be understood as including any single or fused aromatic ring system and include both hetero and other ring systems.
• substituted phenyl as used herein includes any phenyl group having one or more substituents attached to the phenyl ring, at any ring carbon atom of the ring. Where there is more than one substituent on the phenyl ring those substituents may be the same or may be different from one another.
• Electrochemical detection has the potential for very high levels of sensitivity and exhibits a wider linear dynamic range than fluorescence. There is no requirement for samples to be optically clear. There is also less interference from background contaminants (many biological samples auto-fluoresce).
• Electrochemical detection is based on the observation that an electrochemically active marker exhibits different electrochemical characteristics depending on whether or not it is attached to a substrate and on the nature of the substrate. For example, in the case of an electrochemical label attached to an amino acid, the exhibited characteristics will depend not only on the identity of the amino acid but also on whether or not that amino acid residue is incorporated into a peptide or protein, and on the length of any such peptide or protein. Under appropriate circumstances, the electrochemical activity of a marker attached to an amino acid residue can change by a detectable degree following loss of attachment of a single or very few amino acid residues.
• the size and characteristics of a molecule to which an electrochemically active marker is attached influence the observable characteristics of the electrochemical marker. That may occur, for example, by influencing the rate of migration of the marker by diffusion or its rate of migration in response to an electric field.
• Electrochemical activity of a marker may also be influenced by steric effects resulting from the presence of the molecule to which it is linked. For example, steric hindrance may prevent the marker from approaching an electrode and accepting or donating electrons.
• the secondary structure of the peptide may influence the physical properties of the marker. For example, if the marker is attached to an amino acid residue in a peptide such that the structure of the peptide sterically hinders the electrochemically active marker then the signals observable by voltammetry may be reduced. Digestion of the peptide may destroy or release secondary structure elements and thus reduce or abolish the influence of the peptide structure on the marker. Accordingly, digestion of the peptide results in a change, usually an increase, in the electrochemical signal produced by the marker moiety. In a differential pulse voltammetry experiment, the Faradaic current response at a particular applied voltage may increase upon digestion of the peptide.
• the electrochemical characteristics will be influenced by whether or not the nucleotide is incorporated into an oligonucleotide, upon the length of that oligonucleotide, and upon the sequence of the oligonucleotide especially in the vicinity of the point of attachment.
• the information relating to the electrochemically active marker can be obtained by voltammetry or by an amperometric method. Differential pulse voltammetry is particularly suitable.
• the electrochemical detection step may be carried out using one or more electrodes covered by a membrane which is able selectively to exclude molecules based on one or more characteristics, for example, size, charge or hydrophobicity. That may assist in eliminating background noise current arising from, for example, charged species in the solution.
• the two ferrocenyl groups Fc and Fc' are each independently selected from substituted ferrocenyl groups having one or more substituents as defined above with reference to general formula I.
• One or both pentadienyl rings of one or each of the ferrocenyl moieties may be substituted by one or more substituents, the nature and location of which are selected so as to influence in a desired manner the redox characteristics of the ferrocene moiety.
• the pentadienyl rings of the ferrocenyl moiety may further be substituted by any further ring substituent(s) that do not materially reduce the electrochemical sensitivity of the label, or by any further ring substituent(s) that will enhance the electrochemical or other characteristics of the label in any respect.
• Fc and Fc' are the same and each comprise at least one substituent selected from the group consisting of:
• Fc and Fc' are the same and there is as substituent a cyano group located on the respective proximal cyclopentadienyl ring of each of said Fc and Fc' moieties.
• Fc and Fc' are the same and each comprise at least one ring substituent selected from sulfur-containing groups and phosphorus-containing groups.
• a compound with an electrochemical potential value of in excess of 500mV it is believed that compounds of the invention that include sulfur-containing groups or phosphorus-containing groups, especially those in which the sulfur or phosphorus atom is directly boded to a ring carbon of the ferrocenyl, will be advantageous in extending the range of available potential values of such labels, making them useful as labels in electrochemical assays.
• those compounds offer the possibility of use in assays in which the high electrochemical potential value may be valuable, for example in multiplex assays where a range of different labels with differentiable electrochemical potentials are used.
• a sulfur-containing group at least one sulfonyl substituent selected from groups of the formula R 15 S(O) 2 -, wherein R 15 is selected from C1 to C4 branched- or straight-chain alkyl, C1 to C4 haloalkyl, unsubstituted aryl or aryl substituted with one or more substituents selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, and C1 to C4 alkoxy.
• Illustrative alkyl groups R 15 include, for example, methyl, ethyl, propyl, or butyl, especially t-butyl.
• Preferred haloalkyl groups R 15 include, for example, fluoroalkyl groups with one of more fluoro substituents, especially trifluoromethyl.
• R 15 represents unsubstituted C1 to C4 alkyl; or C1 to C4- haloalkyl, for example C1 to C4- fluoroalkyl, especially trifluoromethyl.
• Illustrative aryl groups R 15 include, especially, phenyl, which may be substituted or unsubstituted, with preferred substituents including, for example, halo, unsubstituted alkyl (preferably C1 to C4 alkyl), substituted alkyl (for example haloalkyl), nitro, cyano, alkoxy (for example, C1 to C4 alkoxy, preferably methoxy) and sulfur-containing groups, for example sulfonyl.
• Other illustrative aryl substituents R 15 include heteroaryl groups containing at least one heteroatom selected from oxygen, sulphur and nitrogen.
• R 15 are those of general formula (R 16 ) a -Ar-, or (R 16 ) a -HeAr- in which Ar represents aryl; HeAr represents heteroaryl; R 16 is a substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, alkoxy, and sulphur-containing groups, for example sulfonyl; and a is an integer in the range of from 0 to a number equal to the maximum substitutable ring positions in the aryl, or heteroaryl ring.
• R 15 may represent phenyl substituted by F; Cl; Br; I; unsubstituted C1 to C4 alkyl; C1 to C4 haloalkyl, for example trifluoromethyl; nitro; cyano; methoxy; or sulfur-containing groups, for example sulfonyl.
• R 17 represents C1 to C4 alkyl, which is preferably unsubstituted, for example, methyl, ethyl, propyl, or buty
• Illustrative aryl substituents R 17 include phenyl and heteroaryl groups containing at least one heteroatom selected from oxygen, sulphur and nitrogen, each of which may be unsubstituted or substituted.
• Preferred aryl groups R 17 include those of general formula (R 18 ) b -Ar- , or (R 18 ) b -HeAr- in which Ar is aryl; HeAr is heteroaryl; R 18 is a substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, C1 to C4 alkoxy and sulphur-containing groups, for example sulfonyl; and b is an integer in the range of from 0 to a number equal to the maximum substitutable ring positions in the aryl or heteroaryl ring.
• R 17 may represent phenyl substituted by F, Cl, Br, I, unsubstituted C1 to C4 alkyl, C1 to C4 haloalkyl, for example trifluoromethyl, nitro, cyano, or methoxy.
• Ar represents phenyl and comprises one or more substituents R 18 (which may be the same or different) selected from halo, alkyl, haloalkyl, nitro, cyano, and alkoxy. More preferably, each R 17 is the same and represents phenyl with at least one substituent, especially phenyl with one subsituent in the 4-position.
• R 17 represents branched C1 to C4 alkyl, for example t-butyl.
• the ferrocenyl groups are the same and there is present as a said substituent on each ferrocenyl at least one substituted phenyl group in which the phenyl has at least one substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, C1 to C4 alkoxy, and sulfur-containing radicals, for example, sulfonyl.
• substituents on the phenyl are, for example, fluorine, chlorine, bromine, iodine atoms, nitro, cyano, trifluoromethyl, and methoxy.
• each phenyl has one substituent which may be located in the 4-position, for example, 4-nitrophenyl (wherein the ferrocenyl group is attached to the phenyl group at the 1-position).
• each ferrocenyl at least one heteroaryl group which may be unsubstituted or substituted by at least one substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl and C1 to C4 alkoxy.
• substituents selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl and C1 to C4 alkoxy.
• each ferrocenyl there may be present as a said substituent on each ferrocenyl at least one iodine or chlorine atom.
• substituents include at least one C1 to C6 alkyl silyl substituent, preferably trimethylsilyl.
• each ferrocenyl moiety may optionally be further substituted by at least one additional substituent selected from bromo, fluoro, C1 to C4-alkyl, haloalkyl, and C1 to C4 alkenyl.
• Fc and Fc' may each additionally comprise at least one cyano group substituent on its distal ring.
• the ferrocenyl moieties are identical. That is thought to give a stronger signal.
• the moiety Z may be unsubstituted or substituted.
• Substituents when present, may be for example one or more substituents selected from hydroxy, halo, cyano, amino, and unsubstituted or substituted C1-C4 alkyl, C1-C4 alkenyl, or aryl; wherein in each case optional substituents include without limitation hydroxy, halo, cyano, oxo, amino, ester or amido.
• the moiety Z may, if desired, be interrupted by one, or optionally more than one, atom or moiety selected from -O-, -S-, cycloalkyl, including heterocycloalkyl, -CO-, -CONH-, -NHCO- and -NH- and -NR 1 - in which R 1 is C1 to C4 alkyl.
• cycloalkyl moieties that may be included as interruptions within the moiety Z are cycloalkyl rings with from 5 to 7 ring atoms, especially 6 ring atoms, for example cyclohexyl, piperidinyl, morpholinyl.
• the moieties X and Y which are preferably the same, advantageously have a chain length of from 1 to 6, preferably from 1 to 4 carbon atoms, especially one or two carbon atoms, and more especially one carbon atom.
• the moieties X and Y may each represent an alkylene chain, optionally interrupted by -O-, -S- or -NR 5 - for example -NH-.
• Preferred moieties X and Y include, for example, -CH 2 -, -CH 2 -CH 2 -, -(CH 2 ) 3 -O-CH 2 -, -CH 2 -O-(CH 2 ) 3 -, -(CH 2 ) 3 -O-(CH 2 ) 2 -, and -(CH 2 ) 2 -O-(CH 2 ) 3 -.
• labels according to the present invention may be prepared by reacting two equivalents of a suitable substituted ferrocene carboxaldehyde in a suitable solvent in the presence of a reducing agent.
• the structure of the desired label, including the structure of moiety Z, may be determined by selection of suitable starting materials and/or routine modification of the synthesis method.
• a ferrocene derivative such as 1'-iodo ferrocene carboxaldehyde, 1'-chloro ferrocene carboxaldehyde, 1'-furanyl ferrocene carboxaldehyde or 2- tert -butyl sulphonyl ferrocene carboxaldehyde
• a suitable amine for example, 6-aminohexan-1-ol, glycine or (aminoethoxy)ethanol
• a suitable solvent for example THF
• a reducing agent for example sodium triacetoxyborohydride.
• the resulting di-ferrocenyl glycine derivative may be further modified to generate a desired structure. For example, it may be reacted with oxalyl chloride in dichloromethane then treated with 4-(hydroxymethyl)piperidine to generate a ferrocene-substituted derivative of 2-((di-ferrocenylmethyl)amino)-1-(4-(hydroxymethyl)piperidin-1-yl)ethanone.
• the resulting di-ferrocenyl glycine derivative when glycine is used as the amine, the resulting di-ferrocenyl glycine derivative may be further reacted with oxalyl chloride in dichloromethane then treated with 6-aminohexan-1-ol to generate a ferrocene-substituted derivative of N , N -2-(diferrocenylmethylamino)acetyl-6-aminohexanol (also named N-(6-hydroxylhexyl)-2-((diferrocenylmethyl)amino)-acetamide).
• Suitable methods for synthesis of other compounds according to the invention will be apparent to those skilled in the art in the light of the disclosure herein.
• Linkage to the substrate can be by any suitable linkage, typically by linkage to a substrate side chain.
• the linker group R in the compounds of general formula I is a hydroxyl group or a protected hydroxyl group or a group containing a hydroxyl group or a protected hydroxyl group. It will be appreciated, however, that any other suitable linker group R may be selected having regard to the substrate to which, in use, the compound is to be attached.
• Various synthetic methods have been developed for the derivatisation of protein, peptide or amino acid side chains or protein, peptide or amino acid terminal moieties. For example, lysine residues in a protein may be derivatised by reaction with a succinimidyl ester.
• a maleimide reagent may be used to derivatise cysteine residues.
• An N-hydroxy succinimide ester may be used to derivatise the amino terminus or side chain amino group of a protein or peptide, or an amino moiety of an amino acid.
• Suitable derivatisation methods for nucleotides are also well-known, for example, using a phosphoramidite moiety.
• Labelled substrates according to the invention may be prepared by reaction of a compound according to the invention, optionally after functionalisation to obtain a functionalised labelling compound, with a substrate, for example, with a substrate selected from amino acids, nucleotides (for example oligo deoxyribonucleotides or oligo ribonucleotides), nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates and derivatives of any of those molecules.
• a substrate selected from amino acids, nucleotides (for example oligo deoxyribonucleotides or oligo ribonucleotides), nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates and derivatives of any of those molecules.
• the substrate is a nucleotide or an oligonucleotide.
• the nucleotide may be selected from adenosine, thymidine, guanosine, cytidine or uridine nucleotides.
• the nucleotide, or a nucleotide of the oligonucleotide is attached to the label through a group attached to the ribose or deoxyribose group of the nucleotide, for example in the 2', 3' or 5' position, for example through an oxygen or nitrogen atom.
• the nucleotide is attached at the 3' or 5' position, for example at the 5' position. Linking at other positions is also possible.
• one advantageous way of attaching labels of the invention is by functionalization with phosphoramidite.
• the linking of phosphoramidite groups to oligonucleotides is widely practised in oligonucleotide synthesis and thus methods and conditions for attachment to an oligonucleotide of labels functionalised with phosphoramidite will be well-known and a routine matter to those skilled in the art. Further, it advantageously permits the use of standard oligo manufacturing methods.
• Oligonucleotides suitable for use in an assay in accordance with the invention are advantageously nucleotides having from 2 to 50 nucleotides, more preferably from 2 to 40 nucleotides especially from 15 to 35 nucleotides, with from 18 to 30 nucleotides being especially preferred.
• shorter oligonucleotides may be useful, for example oligonucleotides with from 2 to 14 nucleotides, more preferably from 2 to 10 nucleotides.
• Attachment to proteins may be accomplished in some cases by incubation of the protein and ferrocenyl label together at room temperature in an appropriate buffer solution.
• the label is advantageously to be linked to cysteine or lysine but the substrate sequence does not contain cysteine or lysine at a suitable position the sequence may if desired be mutated to add one or more cysteine or lysine residue either as an additional residue or as a substitution for another residue.
• An alternative method for attachment to proteins may include biotinylation of the labels and use of commercial streptavidinated proteins (or vice versa).
• the substrate may be biotinylated by any standard technique for example by use of a commercially available biotinylation kit.
• Biotinylated substrate will bind to strepavidin or avidin conjugated compounds such as antibodies (which are commercially and widely available).
• Suitable functionalising groups may include, without limitation, succinimidyl ester groups, phosphoramidite groups, maleimide groups, biotin and azide groups. It will be appreciated, however, that there may be used any functionalising group that facilitates attachment of the labelling compound to the substrate to be labelled.
• the invention also provides a method of detecting a nucleic acid (for example RNA or DNA) in a sample comprising the optional step of amplifying the nucleic acid (for example by PCR or another nucleic acid amplification technique) followed by the step of contacting the amplicon (or the nucleic acid) with a complementary nucleic acid probe under conditions to allow hybridization between the probe and amplicon (or the nucleic acid), followed by the step of selectively degrading either hybridized or unhybridized probe (for example by use of single or double strand specific nucleases), wherein said probe is labelled with an electrochemically active compound of the invention and wherein the method provides the step of measuring the electrochemical activity of the compound labelling the probe of wherein said electrochemical activity is dependent either quantitatively or qualitatively on the extent of degradation of the probe.
• a nucleic acid for example RNA or DNA
• the invention also provides a method of detecting an antibody or derivative (which may for example be bound to target antigen in an assay) with an electrochemically active compound of the invention comprising the step of measuring the electrochemical activity of the compound.
• This method can be performed quantitatively or qualitatively.
• the invention also provides methods of diagnosing or monitoring a disease in a subject comprising using a method of the invention in the detection of a protease or a protease inhibitor associated with said disease in a tissue or body fluid of the subject.
• a substrate for the protease can be labelled according to the invention.
• the invention also provides methods of diagnosing or maintaining a disease in a subject comprising using a method of the invention to detect a peptide or protein associated with said disease in a tissue or body fluid of the subject.
• the invention also provides methods of diagnosing or monitoring a disease in a subject comprising using a method of the invention in the detection of a nuclease or a nuclease inhibitor associated with said disease in a tissue or body fluid of the subject.
• the invention provides use of a method of the invention for detecting a disease in a subject.
• the invention also provides methods of detecting a microorganism (in particular, a pathogen or other undesirable organism, for example a food spoilage organism), comprising using a method of the invention.
• a substrate from the microorganism or derived from the pathogen e.g. a nucleic acid amplicon produced using a target nucleic acid sequence in the pathogen
• Detection of the labelled substrate can be used to indicate detection of the microorganism.
• the invention also provides an assay comprising a step which uses a labelled substrate of the invention, optionally in combination with other assay components for example a sample vessel, a container comprising electrodes for electrochemical detection, enzymes suitable for use in the assay or standards and controls.
• Said assay may use more than one different labelled substrate of the invention. If that is the case the presence of different labelled substrates may be differentially detected by labelling them with electrochemical labels of the invention having different electrochemical characteristics (for example different oxidation potentials) thereby permitting the assay to be a multiplex (for example a duplex) assay in which different substrates may be discriminated when present in the same sample vessel.
• Simplex assays are also encompassed by the invention.
• Table 1a sets out certain illustrative formulae of compounds according to the invention which may be used as labels in electrochemical assays in accordance with the invention, and which may be used to make functionalised labelling compounds and labelled substrates according to the invention.
• Table 1a also sets out in the second column illustrative corresponding functionalised labelling compounds according to the invention.
• Tables 1b, 2, 3 and 4 set out general formulae of further illustrative compounds of the invention. Whilst functionalised compounds corresponding to the compounds identified in Tables 1b, 2, 3 and 4 are not shown, it will be appreciated that the compounds shown may be functionalised by addition of any suitable functionalising moiety.
• each ferrocenyl may have more than one substituent, which may be the same or different, and in any ring position. Both ferrocenyl groups preferably have the same substituent(s) in the same positions, i.e. both ferrocenyl groups are the same.
• R 10 represents a radical selected from S-containing groups, P-containing groups, I, Cl, trialkylsilyl, CF 3 , heteroaryl, substituted phenyl; q represents from 1 to 5, for example 1; and W represents (CH 2 ) n where n is from 0 to 6, O, S or NR 20 where R 20 is alkyl, for example C1 to C4 alkyl 2
• R 11 represents a radical selected from S-containing groups, P-containing groups, I, Cl, trialkylsilyl, CF 3 , heteroaryl, substituted phenyl and cyano; r represents from 1 to 5, for example 1; and W represents (CH 2 ) n where n is from 0 to 6, 0, S or NR 20 where R 20 is alkyl, for example C1 to C4 alkyl
• the compounds 6 to 8 above may be functionalised by any suitable method, for example by phosphoramidation analogously to the compounds 1 to 5 shown in Table 1 above.
• Table 2 there are shown general formulae describing certain preferred embodiments of the invention in which each ferrocenyl group is substituted by a sulfonyl group.
• Table 3 shows general formulae describing certain preferred embodiments of the invention in which each ferrocenyl group is substituted by a phosphinyl group.
• the compounds in Tables 2 and 3 may each be functionalised by any suitable method, for example phosphoramidation.
• the present invention encompasses the functionalised analogs of the compounds defined in the Tables 1a, 1b, 2 and 3 as well as labelled substrates derived therefrom. Where present in the formulae 9 to 20 above:
• each distal pentadienyl ring of each ferrocenyl that is, the ring remote from the bond linking the ferrocenyl to the rest of the molecule
• those substituents may be in any position relative to one another.
• ring substituents on either the proximal or the distal ring, it is also possible for both pentadienyl rings of each ferrocenyl to carry one or more substituents.
• incorporation of one or more substituents on each of the ferrocenyl groups can be used to obtain compounds with modified electrochemical characteristics, providing through appropriate substituent selection a suite of compounds from which two or more may be selected for the purpose of multiplex reactions.
• 1'-Furanyl ferrocene carboxaldehyde was synthesised from 1'-iodoferrocene carboxaldehyde, using method adapted from Angewandte Chemie, 2006, 45, 1282 - 1284 .
• 2- tert -Butyl sulphonyl ferrocene carboxaldehyde was obtained from synthesised from dimethylaminomethyl ferrocene using method adapted from Organometallics, 1985, 7, 1297-1302 .
• 6-(Bis((2-formyl)1-methylferrocenyl)amino)hexan-1-ol was synthesised from (+/-)-4-(methoxymethyl)-2-ferrocenyl-1,3-dioxane using a method adapted from Journal of Organic Chemistry, 1997, 62, 6733-6745 .
• Iodoferrocene was synthesised from ferrocene, from an adaptation of the method described in Journal of Organometallic Chemistry, 2011, 696, 1536-1540 , utilising iodine as a suitable electrophile.
• Chloroferrocene prepared from ferrocene using a modified procedure from J.Organomet. Chem., 1996, 512, 219-224 , using hexachloroethane as a chlorinating reagent.
• 6-Aminohexanol, ferrocene, dimethylaminomethyl ferrocene and 4-nitrobenzene boronic acid were obtained from Sigma-Aldrich.
• the electrochemical potential values mentioned hereafter were measured using an electrochemical cell including as background electrolyte an aqueous 100mM solution of sodium chloride, using a printed carbon working electrode, a printed carbon counter electrode and a silver/silver chloride reference electrode, all with silver connectors.
• the electrodes were ink based and were screen printed on to a polymer substrate (for example Mylar) followed by heat curing.
• the sample may be prepared as follows: Ferrocenyl label precursor (2ng) is dissolved in DMSO (1mL). An aliquot of 10 ⁇ L is taken of this solution and is then further diluted in the buffer (500 ⁇ L). Then an aliquot (20 ⁇ E) is applied to the screen printed electrode to run the electrochemical scan.
• An illustrative form of suitable cell is described and shown schematically in WO2012/085591 .
• the electrochemical potential was measured and found to be 442 mV.
• the electrochemical potential was measured and found to be 437 mV.
• the electrochemical potential was measured and determined to be 339 mV.
• the electrochemical potential (DPV) was measured and found to be 512 mV.
• the electrochemical potential (DPV) was measured and found to be 452 mV.
• 6-(bis((2-formyl)1-methylferrocenyl)amino)hexan-1-ol (leq) is dissolved in ethanol and treated with hydroxylamine hydrochloride (5 eq) and sodium acetate (5 eq). The resulting suspension is then heated at reflux for 18 hrs. After this time the reaction is allowed to cool to room temperature and concentrated in vacuo. The solid residue is then partitioned between chloroform and NaHCO 3 (sat). The organic layer is separated and dried over Na 2 SO 4 , filtered and concentrated in vacuo to give the corresponding oxime.
• the oxime is then taken up in dry THF and treated with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP - 2 eq) and stirred for 5 mins. Then 1,8-diazabicyclo[5.4.0]undec-7-ene (2.3 eq) is added. The solution is then stirred for 90 mins. The reaction is then diluted with EtOAc and washed with water and brine (sat). The organic phase is dried over MgSO 4 , filtered and then concentrated in vacuo.
• BOP - 2 eq (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
• the 2-tert-butylsulfidyl-ferrocene carboxaldehyde (237 mg, 0.78 mmol) was placed in a round bottomed flask with 6-amino-hexanol (46 mg, 0.39 mmol) and dissolved in dry THF (5 cm 3 ).
• the suspension was then treated with sodium trisacetoxyborohydride (322 mg, 1.96 mmol).
• the flask was equipped with a condenser and refluxed overnight. After this time the flask was allowed to cool to room temperature.
• the reaction was quenched by addition of NaHCO 3 (sat) (10 cm 3 ). Organics were separated and the aqueous layer back extracted with EtOAc (3 ⁇ 5 cm 3 ).
• the ferrocenyl derivative shown as a starting material in the above reaction scheme is illustrative, and may be replaced by a molar equivalent of any of the compounds made in Examples 1 to 9 above.
• N,N-diisopropylethylamine (0.4mL, 8.4mmol) was added to a stirred solution of the ferrocene derivative (2.1mmol) in dry THF (25mL) under a nitrogen atmosphere.
• 2-cyanoethyldiisopropylchlorophosphoramidite (0.2ml, 3.15mmol) was added dropwise and the resulting mixture was stirred for 15mins.
• MilliQ filtered water 200mL was added and the solution was stirred for a further 30mins.
• Ethyl Acetate - Triethylamine (1:1, 25mL) was added, a precipitate formed.
• the labels of the invention are attached to a peptide by attachment of the label to a free amine of, for example, a lysine residue in the peptide. Attachment may be accomplished conventional techniques including functionalisation of the labelling compound to form an active NHS ester and reaction of the functionalised ester with the free amine group of the peptide.
• a biotin molecule is coupled to a label, for example a label as made in any of Examples 1 to 9.
• the biotinylation can be carried out in an automated oligonucleotide synthesiser or using standard laboratory conditions by reaction of ferrocenyl phosphoramidite label with N-hydroxysuccinimide (NHS) esters of biotin.
• NHS N-hydroxysuccinimide
• Paramagnetic treptavidin particles are washed x 3 (phosphate buffer) and mixed with biotinylated label, followed by incubation for 1 hour at room temperature with mixing.
• the particles are washed x 2 (phosphate buffer) and washed x 1 (PCR buffer). They are resuspended in final buffer (PCR buffer). Following each wash step the supernatants are tested for electrochemical signal, and if necessary washing is repeated until the supernatants show no indication of free electrochemical label.
• These particles are assayed at a range of concentrations to validate that the observed electrochemical signal is attributable to the label coupled to the magnetic particles, using magnetic capture of the particles and resuspension in a range of buffer volumes.
• the data in the above table shows that the compounds of Examples 1 to 5, 8 and 9 provide useful electrochemically active labels.
• the labels may be used to provide an electrochemical signal within a desired range of values. They may be useful as alternative labels to other labelling compounds with similar potential values, for example, where those other labelling compounds have disadvantageous properties in the assay in question, for example, incompatibility with impurities or other components present in the assay or incompatibility with the measurement conditions, any of which could affect measurement sensitivity.
• they may be used with one or more other labels in a multiplex assay in which more than one label is present to provide two or more determinations in a single sample, the use of two or more labels with different electrochemical properties in those circumstances permitting effective distinction between measurements relating to the respective species to be determined.

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